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Department of Endocrinology and Metabolism, University of Stellenbosch Medical School, Tygerberg 7505, Cape Town, South Africa
Address all correspondence and requests for reprints to: Dr. P. A. Hulley, Department of Endocrinology and Metabolism, University of Stellenbosch, Medical School, P.O. Box 19063, Tygerberg 7505, South Africa. E-mail: phul{at}maties.sun.ac.za
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Abstract |
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The dexamethasone-induced decline in preosteoblast proliferation correlated with a 30–40% reduction in protein kinase C/TPA-stimulated mitogen-activated protein kinase (MAPK) activity. These steroid effects only became evident after 6–24 h treatment, implying that dexamethasone acts on de novo synthesis of proteins. Because MAPK is inactivated by dephosphorylation of tyrosine and threonine residues, cells were treated concomitantly for 24 h with dexamethasone and inhibitors of tyrosine (sodium orthovanadate) and/or serine/threonine phosphatases (sodium fluoride). MAPK activity and cell proliferation were restored when MBA-15.4 cells were treated with vanadate, suggesting that dexamethasone up-regulates tyrosine phosphatase activity. Inactivation of serine/threonine phosphatases with sodium fluoride had no effect. Inhibition of the PKA pathway (which is growth inhibitory in mature osteoblasts) with H-89 did not reverse the effects of dexamethasone. Pretreatment with dexamethasone inhibited both peak- and extended activation plateau-phases of MAPK activity. Both phases were fully restored by pretreatment with vanadate, implicating more than one tyrosine phosphatase. Cycloheximide, alone or in combination with dexamethasone, prevented drop-off from plateau to basal levels, suggesting that an inducible dual-specificity phosphatase regulates the plateau-phase.
We conclude that dexamethasone may inhibit preosteoblast growth via a novel tyrosine phosphatase pathway.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Glucocorticoids primarily act by up- or down-regulating gene
transcription, and in the case of osteoblasts, multiple proteins are
likely to be involved (reviewed in Refs. 2 and 3). Growth of bone is
impaired by down-regulation of gonadotropin and sex steroid production
(4) and also by down-regulation of local osteoblast growth factors such
as insulin-like growth factor (IGF)-I, IGF-II, and transforming growth
factor-ß (reviewed in Ref.2). Osteoblasts of different ages are
affected by steroids in different ways. Preosteoblasts are driven to
stop dividing and differentiate, expressing mature bone markers such as
alkaline phosphatase and osteocalcin (5). However, mature osteoblast
function is also impaired by steroid treatment, with an apparent
decrease of osteocalcin and 
1(I)-collagen transcription
and an increase in production of collagenase 3, which degrades type-I
collagen (2). These changes result in a bone cell population that, in
spite of the initial surge of steroid-induced differentiation, is
deficient in young, proliferating osteoblasts and in which the mature
osteoblasts are functioning poorly.
Whereas the inhibition of osteoblast proliferation caused by steroid therapy may, in part, be caused by down-regulation of external growth signals such as circulating hormones or local growth factors and their receptors, we were interested to see whether glucocorticoids might exert some of their inhibitory effects directly on intracellular mitogenic signaling pathways. The best characterized mitogenic pathway is the highly conserved extracellular signal regulated protein kinase (ERK) or mitogen-activated protein kinase (MAPK) cascade (reviewed in Refs. 6–8). When mitogens such as insulin or growth factors bind to their receptor protein tyrosine kinases (RPTK) on the cell membrane, the grb2 adapter molecule recruits Sos, a guanine nucleotide exchange factor, to form a complex with the RPTK. Sos promotes conversion of ras-GDP to ras-GTP, and activated ras binds to Raf-1 and positions it next to its activator kinase (possibly the RPTK) in the plasma membrane. At the same time, binding of growth factor to the RPTK mediates activation of protein kinase C, which phosphorylates Raf-1, and together the actions of ras and PKC produce fully active Raf-1. Raf-1 serine-phosphorylates the dual-specificity kinase, MAPK kinase, or MEK (6, 8). MEK then phosphorylates ERK-1 and 2 (ERK-1/2) on both threonine and tyrosine in a TXY motif common to MAP kinases (7, 9). Phosphorylation of both residues is necessary for activation of ERK-1/2 (9) and for its subsequent translocation to the nucleus (6, 7, 10). ERK-1/2 phosphorylates serine or threonine residues on a wide range of proteins, including transcription factors such as c-jun, c-fos, and TCF/Elk-1.
Dexamethasone induces a general reduction of tyrosine phosphorylation, both in bone cells (our observation) and in other cell types (11, 12). This could be brought about by transcriptional regulation leading to an inhibition of kinase activity and/or stimulation of phosphatase activity. One mechanism established by our group (13) and others (14, 15) by which MAPK activity is inhibited in bone is by PTH-induced activation of protein kinase A. PKA inhibits Raf-1 from binding to ras by a specific phosphorylation in its Ras-binding domain, and thereby blocks the MAPK cascade (reviewed in Ref.8). Steroids have recently been shown to inhibit the MAPK pathway at the level of Raf-1 in mast cells, and a reduction in the phosphorylation of Raf-1, MEK, and ERK-1/2 is apparent (16). It is not known whether this dephosphorylation is brought about by PKA, phosphatase action, or by another unknown mechanism. Phosphatases that act to inhibit cell proliferation can be induced by dexamethasone as illustrated by the up-regulation of the hemopoietic cell phosphatase SHP-1 (formerly known as HCP-1 or SH-PTP1c), which associates with activated receptors through its SH2 domains and inhibits growth factor-induced tyrosine phosphorylation and mitogenesis in rat AR42J pancreatic cells in vitro (17).
We have found that characteristically more mature osteoblast cell lines, such as UMR-106 and Rob-C26 cells, respond to dexamethasone treatment by differentiating but not with any marked effect on proliferation. The same limitation holds true for primary bone cell cultures, which are inevitably heterogeneous with respect to osteoblast age and state of differentiation. However, a preosteoblastic, bone marrow stromal cell-line, MBA-15.4, was found to be severely growth-inhibited by dexamethasone (18). This cell line expresses osteoblastic markers such as alkaline phosphatase and collagen type I in vitro and can be induced to produce bone in vivo (19). We therefore used MBA-15.4 cells to investigate the possibility that glucocorticoids might inhibit proliferation of preosteoblasts by inducing synthesis of phosphatase enzymes capable of inactivating the MAPK cascade.
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Materials and Methods |
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Polyclonal antibody against MAPK was from Upstate Biochemicals Inc. (Lake Placid, NY), and those for MEK and Raf-1 came from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Secondary alkaline phosphatase- and peroxidase-coupled antirabbit antibodies were from Boehringer Mannheim (Mannheim, Germany) and Amersham, respectively. BCIP and NBT were also from Boehringer Mannheim. All other chemicals, including tissue culture media, O-tetradecanoylphorbol 13-acetate (TPA), forskolin and human PTH fragment, hPTH (1–34), were of the highest grade, obtained from Sigma.
Cell culture
MBA 15.4 mouse bone marrow stromal cells were a kind gift from
Professor S. Wientroub (Tel Aviv University, Israel). They express
osteoblastic markers such as alkaline phosphatase and collagen type I,
but very low levels of PTH receptors in vitro, and can be
induced to produce bone in vivo (18, 19). Rob-C26 cells were
likewise donated by Professor A. J. Kahn (University of
California, San Francisco, CA).
MBA 15.4 cells were grown in bicarbonate-buffered DMEM with 10%
heat-inactivated FCS, 100 U/ml penicillin, and 100 µg/ml
streptomycin. Rob-C26 cells (21) were cultured in -MEM containing
5% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. For
experiments, 70% confluent cells were trypsinized with 0.05% trypsin
in 0.01% EDTA and seeded in 24-well plates (growth studies), or 100-mm
culture dishes (MAPK assays) at 5 x 103 cells per
cm2. Medium was changed to DMEM (or EMEM for Rob-C26 cells)
with 2% FCS 24 h before all experiments to serum-starve the
cells. Neither cell line tolerated serum-free culture.
Cell proliferation
DNA synthesis was assessed by measurement of
[3H]thymidine incorporation into acid-insoluble material,
as previously described (22). Briefly, MBA-15.4 cells were grown in
24-well plates in DMEM with 10% FCS. To investigate the effect of
dexamethasone on proliferation of cells growing in different
concentrations of FCS, the medium in 50% confluent cultures was
replaced with medium containing 0.5%, 10% or 20% FCS, to which
10-6 M dexamethasone was added for periods of
18, 24, or 48 h. In all other proliferation studies, cells with
synchronous growth cycles were produced by replacing medium of
subconfluent cells with DMEM plus 2% FCS 24 h before experiments,
whereafter medium was replaced with DMEM plus 2% FCS containing the
experimental compounds for various periods up to 24 h. In every
experiment, 2 µCi/well [3H]thymidine was added to the
medium for the last 2 h of incubation. The incubation was stopped
on ice, the medium removed, and the cells washed twice with PBS before
being quick-frozen at -70 C. After lysis with 0.1 M
NaOH–0.1% SDS at room temperature, protein and DNA were precipitated
with cold TCA at a final concentration of 12% overnight at 4 C. The
pellet was washed once with cold 10% TCA before it was dissolved in
0.1 M NaOH. Aliquots were counted in Ready Gel aqueous
scintillation fluid (Beckman, Fullerton, CA) to obtain a measure of
[3H]-thymidine incorporation.
MAPK assay
This assay was performed essentially after the published method
(23). At ±70% confluence, cells in 100-mm plates in 10 ml DMEM with
2% FCS were incubated with 100 ng/ml TPA (1.6 x
10-6 M), 20% FCS, 10-8–
10-4 M dexamethasone, 10-5 or
10-6 M sodium fluoride, 5 x
10-6 sodium orthovanadate, or 10-6
M cycloheximide for the required times at 37 C. A range of
concentrations was tested for each substance used to establish the most
effective, nontoxic dose. When the PKA inhibitor H-89 was used, it was
added to a final concentration of 20 µM in 0.1% DMSO for
30 min before the cells were stimulated (24). Stimulation of the cells
was terminated by placing plates on ice and removing the medium. Cells
were washed twice with ice-cold 20 mM Tris-HCl at pH 7.4
containing 1 mM EGTA, 10 µM
Na3VO4, 50 mM NaF, and 1
mM PMSF, after which the cells were scraped in 0.4 ml of
the above buffer to which 20 mM paranitrophenyl phosphate
(pNPP), 2 mM DTT and 1 mM levamisole had been
added (lysis buffer). The cell membranes were disrupted by sonication
(1 x 10-sec burst) and centrifuged in a microfuge for 10 min at 4
C. The pooled lysate from duplicate treatments was added to 400 µl of
a 50% DEAE Sephacel suspension, previously equilibrated in the lysis
buffer. Following a 10-min equilibration, the unbound material was
removed after centrifugation of the slurry, the beads washed twice with
1 ml of the above lysis buffer containing 100 mM NaCl, and
the bound proteins were eluted in 400 µl of the lysis buffer
containing 350 mM NaCl. The total protein content of the
eluate was determined by the method of Bradford (25), using BSA as a
standard.
Further batch purification of the DEAE-eluted proteins was carried out
on hydrophobic interaction matrices in the first experiments performed
(see Figs. 3
, A and C, and 6A), but this step was discontinued because
comparable and more rapid results could be obtained with DEAE
purification only. DEAE eluates (100–150 µg protein) were adjusted
to 1.2 M NaCl and added to 200 µl of a 50% slurry of TSK
butyl-sepharose in the same buffer. After equilibration, the beads were
washed with the same buffer containing 0.4 M NaCl before
eluting in 120 µl of the same buffer without NaCl and NaF but
containing 10 mM pNPP and 50% ethylene glycol. Constant
volumes (50 µl) of these eluates were used in assays.
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Western blotting
Cell lysates were prepared by sonication in RIPA buffer,
containing 0.1% Triton-X 100, phosphatase inhibitors (100
µM Na3VO4, 50 mM NaF,
20 mM pNPP and 1 mM levamisole) and protease
inhibitors (1 mM PMSF, 1 µg/ml each of aprotinin,
leupeptin, and pepstatin). Protein concentrations were determined using
the BCA method, which is detergent independent (Pierce Scientific,
Rockford, IL), and equal-protein samples (
1 µg/µl) were mixed
with 3 x Laemmli sample buffer, containing ß-mercaptoethanol,
and placed in a boiling water bath for 5 min. After centrifugation at
14,000 x g for 1 min, proteins were separated by
SDS-PAGE on a 12% acrylamide gel.
Western blots of gels were analyzed for Raf-1, MEK, and MAPK using rabbit polyclonal antibodies and alkaline phosphatase color reaction.
Statistical analysis
All results obtained from MAPK and cell proliferation assays are
expressed as the mean ± SEM (see exception in Fig. 4, mean ± SD). The significance of differences was
calculated by the Student’s one-tailed unpaired t test. A
difference between treatment groups was considered significant at
P < 0.05.
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Results |
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). This
dropped by 30–40% after 10 min and remained at this level for 1–2 h,
returning to basal levels after 4 h (Fig. 1A). Dexamethasone has
been reported to decrease proliferation of MBA-15.4 cells and induce
differentiation into a more mature phenotype (18). We found that this
decrease in proliferation was parallelled by a decrease in MAPK
activity. Pretreatment of MBA 15.4 preosteoblasts for 24 h with
10-6 M dexamethasone inhibited PKC-stimulated
MAPK activity by 30–40% at 5-min peak stimulation and also by
10–20% along the MAPK activity shoulder, from 10 min to 4 h,
after which activity returned to baseline levels (Fig. 1A). This effect
was observed for doses of dexamethasone between
10-8–10-4 M and for a treatment
period of 6–24 h (results not shown for all doses and times).
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and 3A) and shoulder
(Fig. 1B) phases. However, sodium fluoride not only failed to restore
dexamethasone-impaired MAPK activity but actually inhibited it further.
Sodium orthovanadate partially reversed the inhibitory effects of
sodium fluoride on MAPK activity (Fig. 3A). These results suggest that
tyrosine but not serine/threonine phosphatases play a central role in
the down-regulation of mitogen-stimulated MAPK activity.
Because sodium orthovanadate inhibits tyrosine phosphatases and thereby
blocks tyrosine dephosphorylation, it should have an immediate effect
on MAPK activity. We therefore compared pretreatment with vanadate for
10 min before TPA stimulation with pretreatment for 24 h.
Simultaneous addition of vanadate and TPA was not possible because
vanadate alone gives a brief, 1.5- to 2-fold stimulation of MAPK
activity that is over within 10 min. By pretreating 10 min before the
TPA stimulation, we avoided a potential overlap of effects.
Pretreatment with sodium orthovanadate for either 10 min or 24 h
before TPA stimulation completely restored the dexamethasone-induced
deficit in MAPK activation (Fig. 3B). This strongly suggests that the
initial peak of MAPK activity is down-regulated by a tyrosine
phosphatase that is already present in the cells and is up-regulated by
pretreatment with dexamethasone. A more mature rat osteoblast cell
line, Rob-C26 (21), showed no decrease in MAPK activity after treatment
with dexamethasone (Fig. 3C), in accordance with previous reports using
characteristically more differentiated cell lines (2, 5, 18).
Cell proliferation is the biological end-point of the MAPK cascade.
Treatment of MBA-15.4 cells for 48 h with dexamethasone inhibited
cell proliferation by an average of 64%, whether the cells were
growing in the presence of 0.5%, 10% or 20% FCS (Fig. 4, A–C). It was not possible to maintain
live cultures in the complete absence of growth factor-containing
serum. MBA-15.4 cells growing slowly (11,000 cpm/well tritiated
thymidine incorporation) in low serum (0.5% FCS) medium exhibited a
25% decrease in proliferation after 18 h of dexamethasone
treatment, and this dropped further to a 56% decrease after 48 h
of treatment (Fig. 4A). Rapidly growing cells (40 000 cpm/well
tritiated thymidine incorporation) in serum-rich (10% FCS) medium were
more severely affected, with proliferation dropping by 57% after
18 h of dexamethasone treatment and reaching 72% inhibition by
48 h. Growth in 20% FCS was faster than in 10% FCS but showed
very similar responses to dexamethasone.
When cell growth in low serum medium was directly stimulated with TPA,
dexamethasone caused an average decrease in proliferation of 35% by
24 h (Fig. 5). Concomitant treatment
with 1 or 3 doses of sodium orthovanadate over the course of 24 h
restored proliferation to between 80–86% of TPA-stimulated levels.
The increased proliferation seen with vanadate alone is the cumulative
effect of 24 h of treatment, and this is not seen in Fig. 3A
because there is no activation of MAPK activity detectable 24 h
after treatment with vanadate alone. Sodium fluoride in our hands had
no consistent effect on bone cell proliferation (not shown). These
results suggest that dexamethasone may be directly or indirectly
inducing the activity of a tyrosine phosphatase and thereby inhibiting
MAP kinase activity and osteoblast proliferation.
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), suggesting that dexamethasone is not
acting via the cAMP-PKA proliferation control pathway. In fact, an even
greater reduction of MAPK activity was observed upon treatment with
H-89. This could be explained if the role of cAMP/PKA is reversed in
very young bone cells, and activates mitosis rather than inhibiting it.
To check this, we treated cells with PTH and also with forskolin which
generates cAMP. There was no stimulation of cell proliferation as
determined by tritiated thymidine incorporation (Fig. 6B). The reason
for the observed drop in MAPK activity upon treatment of MBA 15.4 cells
with H-89 remains uncertain. It is not the result of H-89 toxicity,
because all substances used in this study were tested for toxicity
using cell proliferation as an assay. H-89 was used at 20
µM, a concentration that did not inhibit MAPK or PKC
activity in UMR-106 cells in earlier experiments (13), and at which
concentration it is an approximately 3,000-fold less potent inhibitor
of PKC than PKA (24). It therefore seems unlikely that the large
decrease in MAPK activity seen in this study would be the result of
nonspecific inhibition of PKC.
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),
suggesting that an inducible, immediate early gene phosphatase controls
the shoulder of TPA-stimulated MAPK activity in bone cells.
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). Although this could mean that dexamethasone and
cycloheximide both act on the same inducible phosphatase, the
experiment was not conclusive. It is possible that cycloheximide is
simply overriding the effect of dexamethasone. It was not possible to
simultaneously co-treat with dexamethasone and cycloheximide for the
full treatment period because cycloheximide was toxic for MBA 15.4
cells after 8 h and thus only permitted a 3-h pretreatment plus
4 h TPA stimulation. Because dexamethasone only started to take
effect after 6 h of treatment before TPA stimulation, total
overlap of cycloheximide and dexamethasone treatments was not feasible.
However, a 3-h pretreatment is widely used to preferentially inhibit
immediate early gene synthesis. Cycloheximide, alone or in combination
with dexamethasone, had no effect on peak MAPK activity, in agreement
with the activation dynamics of inducible phosphatases (28, 30). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MAPK requires phosphorylation on both tyrosine and threonine residues (7, 9) to be activated and is unique in that it can be activated by a single dual-specificity kinase (MEK). Phosphorylation of tyrosine precedes threonine phosphorylation, and tyrosine-phosphorylated ERK-2 has a higher affinity for MEK, indicating that this may be a rate-limiting step (31). We therefore investigated whether inhibition of MAPK activity by tyrosine and/or serine/threonine phosphatases was regulated by dexamethasone. Sodium orthovanadate (tyrosine phosphatase inhibitor) completely reversed the effect of dexamethasone on MAPK activation and substantially restored cell proliferation, whereas sodium fluoride (serine-threonine inhibitor) was ineffective. This suggests that tyrosine phosphatases play a key role in the control of MAPK activity and cell proliferation in osteoblasts.
Protein tyrosine phosphatases are frequently up-regulated in association with decreased cell proliferation and terminal differentiation. An example is the differentiation of PC12 cells into neuron-like cells with extended neuritic processes under the influence of NGF and bFGF. This process is reversed by treatment with sodium orthovanadate, or the related cations, molybdenum and zinc (32, 33) and appears to involve the tyrosine phosphatase LAR (leukocyte antigen related) (34). Similarly, SHP-1 (SHPTP-1c) is critical for normal development of multiple hemopoietic lineages, as demonstrated by defects in motheaten mice, which have a loss-of-function mutation in SHP-1 (35). SHP-1 levels also increase in differentiating pancreatic cells, and here both differentiation and SHP-1 transcription are under the control of glucocorticoids such as dexamethasone (17). It would therefore be interesting to investigate the role of SHP-1 in osteoblasts.
The lack of effect of sodium fluoride in our experiments was
unexpected, given that sodium fluoride is one of the very few
bone-forming agents in current clinical use (2, 36). It reportedly acts
by increasing the number of osteoblasts (37) and must therefore impinge
on the MAPK-mitogenic pathway at some level, although precisely where
is the subject of some debate. Fluoride is classically described as an
inhibitor of serine/threonine but not tyrosine phosphatases (39, 40),
an exception being the acid phosphatase found in bone cells
(41, 42). In our hands, sodium fluoride is not a reliable in
vitro mitogen for UMR-106, Rob C-26, or MBA 15.4 and 15.6 cell
lines, and actually reduces MAPK activation (Fig. 3A; see also Refs.
38, 43, 44). This does not deny the clinical benefits of fluoride
therapy but does suggest that fluoride may be stimulating MAPK and bone
growth more indirectly, perhaps via pertussis toxin-sensitive
Gi proteins (39). It is obvious that the biochemical
functions of fluoride remain controversial, and it would be interesting
to use other subtype specific inhibitors such as okadaic acid,
calyculin-A, and cyclosporin A (reviewed in Refs. 45 and 46) to
investigate the role of serine/threonine phosphatases in the control of
osteoblast MAPK activity more closely.
Down-regulation of osteoblast growth by the PTH-PKA pathway has been well described in UMR 106 (13, 14) and MC3T3-E1 osteoblasts (15), both of which are characteristically mature osteoblast cell lines, expressing osteocalcin, alkaline phosphatase, and PTH receptors. MBA 15.4 osteoblasts express low levels of PTH receptor and have a negligible PTH response, in keeping with their relatively undifferentiated state (18). Nevertheless, we used the specific PKA blocker, H-89, to investigate whether dexamethasone might be inhibiting MAPK activity by means of this established mechanism. Blockade of PKA failed to increase MAPK activity in MBA 15.4 cells, whereas in UMR 106 cells, it reversed the inhibition of MAPK activity brought about by PTH and cAMP stimulation (13, 14). Neither PTH nor forskolin elicited any proliferative response from the cells, and we therefore assume that the PTH pathway is not active in immature osteoblasts and comes into play once the cells move from the proliferating to the differentiating state.
MAPK activity is central to both cell differentiation and proliferation processes and a variety of activation profiles have been reported, depending on the cell type or activating ligand (28). Mitogenic stimulation produces a rapid peak in MAPK activity (3–5 min), which is followed by an immediate drop in MAPK peak activity in all cells. With some ligands activity decreases rapidly back to basal levels, giving a transient activation profile, whereas with other ligands, there is an initial drop-off of peak activity, followed by an extended activation plateau or shoulder. Drop-off of peak activity has been reported in a variety of cell types to be governed by the serine/threonine phosphatase, PP2A, (9, 28, 47), and/or a tyrosine phosphatase such as SHP-1/2, CD45, or PTP1B (9, 27). These are constantly present in the cell and would therefore be immediately available to inhibit MAPK peak activity, without needing mitogen-stimulated de novo protein synthesis. Our results demonstrate that inhibition of tyrosine phosphatases with sodium orthovanadate fully restored both peak and shoulder phases of dexamethasone-inhibited MAPK activity, whereas sodium fluoride was ineffective. This suggests that tyrosine but not serine/threonine phosphatases are involved in these processes in osteoblasts. The identity of the tyrosine phosphatase/s remains to be elucidated, but our results suggest that they should be substantially up-regulated by a 24-h pretreatment with dexamethasone. Vanadate was able to reverse the effects of dexamethasone even when only added 10 min before TPA stimulation, which further suggests that dexamethasone pretreatment, directly or indirectly, up-regulates tyrosine phosphatase activity in MBA 15.4 preosteoblasts and that inhibition of tyrosine phosphatases is sufficient to rapidly restore normal MAPK activity.
Inactivation of MAPK can also be achieved by dual-specificity, tyrosine/threonine phosphatases, such as MKP-1 (28, 29). However, these phosphatases are immediate early gene products whose transcription is activated by the same mitogen that triggers the MAPK cascade, and which therefore only start to decrease MAPK activity after 30–60 min (28). These inducible phosphatases are not capable of the instantaneous down-regulation of MAPK peak activity but come into play during down-regulation of the shoulder. The only available means of demonstrating the presence of dual-specificity phosphatases as opposed to tyrosine phosphatases at present is the use of protein synthesis inhibitors such as cycloheximide immediately before mitogen stimulation (28, 29). This only inhibits synthesis of the inducible immediate early genes and not ordinary tyrosine phosphatases. Although such a cycloheximide- and vanadate-sensitive inducible phosphatase appears to control the activation shoulder of MAPK activity in our MBA 15.4 osteoblasts stimulated via PKC and in 3T3 fibroblasts stimulated with EGF (28), its induction is not rapid enough to account for the drop in peak MAPK activity seen after 5 min and in fact, rapid deactivation of MAPK has been shown to happen independently of the induction of MKP-1 dual-specificity phosphatase (28, 29). Even though cycloheximide was added 3 h before MAPK stimulation, unlike vanadate, it was unable to repair the dexamethasone-induced decrease in peak MAPK activity, which suggests that there are both inducible and noninducible tyrosine phosphatases acting in the down-regulation of MAPK activity. It would be interesting to examine the expression and regulation of the MKP dual-specificity phosphatase family in MBA 15.4 preosteoblasts. The recently characterized MKP-2 (also called TYP-1) is mitogenically induced, like MKP-1, but has RNA levels peaking between 2 and 4 h and protein levels between 4 and 8 h after mitogenic stimulation (48). The dynamics of this vanadate- and cycloheximide-sensitive phosphatase fit our extended MAPK activation curve better than the more transient MKP-1 induction and activity and imply that there are likely to be several types of dual-specificity phosphatase with different tissue distributions and dynamics.
Sustained activation of MAPK for more than 15–20 min appears to be required for translocation to the nucleus (reviewed in Ref.7), and this correlates with differentiation in PC12 cells (7, 49) and proliferation in fibroblasts and osteoblasts (13, 14, 50). Specificity of signaling can therefore be achieved using the same intracellular pathway but different ligands and cell types (28). For example, EGF treatment of Swiss 3T3 fibroblasts induces sustained MAPK activity for over 2 h, whereas in 3T3-L1 adipocytes and PC12 chromaffin cells, EGF only stimulates a transient peak of activity lasting 10–15 min. On the other hand, sustained activation can be induced by serum in PAE cells and NGF in PC12 cells. The function of transient MAPK stimulation is unclear, although because MAPK can phosphorylate the EGF receptor, Raf-1 and MEK (6), it does not necessarily have to translocate to the nucleus to affect cell processes. In fact, transient activation of MAPK in PC12 cells is reported to increase proliferation (7, 10).
The generation of peak vs. shoulder phases of MAPK activation presumably involves physical, cytoskeletally mediated translocation of the MAPK molecules away from membrane-associated phosphatases. In this way, the transition from activated peak to shoulder could be explained in our system by a membrane-associated tyrosine phosphatase acting to drop peak MAPK activity, whereas a proportion of MAPK molecules are translocated to the nucleus, out of reach of the tyrosine phosphatase, but susceptible to degradation by inducible dual-specificity phosphatases that would start to be transcribed 30–60 min after mitogenic stimulation.
In conclusion, our results using the characteristically immature MBA-15.4 cell line indicate that the negative effects of glucocorticoids such as dexamethasone on bone growth are at least in part mediated by protein tyrosine phosphatase inhibition of MAPK and cell proliferation. Sodium orthovanadate is able to rapidly and effectively reverse this process, and because it is reported to have few side effects in the treatment of diseases such as diabetes (51, 52), it would be interesting to establish whether these results hold true in normal bone cells and whether vanadium supplements have any bone-building effects in steroid-treated animals.
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Acknowledgments |
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Footnotes |
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Received September 19, 1997.
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References |
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